Lipoic Acid Attenuates Aroclor 1260-Induced Hepatotoxicity in Adult Rats Hamdy A. A. Aly,1,2 Ahmed M. Mansour,2 Memy H. Hassan,2,3 Mohamed F. Abd-Ellah2 1
Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
2
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
3
Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, El-Madinah El-Munaworah, Saudi Arabia
Received 29 May 2014; revised 5 December 2014; accepted 7 December 2014 ABSTRACT: The present study was aimed to investigate the mechanistic aspect of Aroclor 1260-induced hepatotoxicity and its protection by lipoic acid. The adult male Albino rats were divided into six groups. Group I served as control. Group II received lipoic acid (35 mg/kg/day). Aroclor 1260 was given to rats by oral gavage at doses 20, 40, or 60 mg/kg/day (Groups III, IV, and V, respectively). Group VI was pretreated with lipoic acid (35 mg/kg/day) 24 h before Aroclor 1260 (40 mg/kg/day). Treatment in all groups was continued for further 15 consecutive days. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase activities and total bilirubin, total cholesterol, and triglycerides were significantly increased while total protein, total albumin, and high-density lipoprotein were significantly decreased. Hydrogen peroxide production and lipid peroxidation were significantly increased while superoxide dismutase and catalase activities and reduced glutathione (GSH) content was significantly decreased in liver. Caspase-3 & -9 activities were significantly increased in liver. Lipoic acid pretreatment significantly reverted all these abnormalities toward their normal levels. In conclusion, Aroclor 1260 induced liver dysfunction, at least in part, by induction of oxidative stress. Apoptotic effect of hepatic cells is involved in Aroclor 1260-induced liver injury. Lipoic acid could protect rats against AroC 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. clor 1260-induced hepatotoxicity. V Keywords: liver; Aroclor 1260; oxidative stress; apoptosis; lipoic acid
INTRODUCTION Polychlorinated biphenyls (PCBs) are ubiquitous, persistent organic pollutants and are a particularly problematic group of environmental contaminants. They comprise a class of 209 individual congeners which are numbered from an order Correspondence to: H. A. A. Aly; e-mail: [email protected] Contract grant sponsor: Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah. Contract grant number: 244/166/1433. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22101
of low to high chlorine substitutions (Mills et al., 2007). Due to their high lipophilicity they are mainly found in non-polar matrices such as lipids and adipose tissue or are attached to particulate matter such as sediments and dust (Giesy and Kannan, 1998). PCBs have been widely used in various applications, including dielectric fluids for capacitors and transformers, paints, adhesives, and pesticides because of their chemical and physical stability. Therefore, they have a strong tendency to bioaccumulate in our ecosystem (Yum et al., 2010). Their resistance to breakdown and their lipophilicity allows them to biomagnify in the food chain and persist in the environment (Hansen, 1987). PCBs are also well known as strong endocrine disruptors, acting as ligands
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for steroid hormone receptors, such as the estrogen receptor (Yum et al., 2010). A number of these have structural and toxicological similarities with “dioxins” and elicit their effects via binding to the aryl hydrocarbon receptor (Roos et al., 2011). It is well known that a number of PCBs are potent liver toxicants (Safe, 1994). Arochlor 1260 shows the highest average percent weight of the chlorine and showing the lowest degradation rate by microorganism among PCB congeners (Yadav et al., 1995). Dechlorination of PCBs has been known to reduce the potential risk of PCB exposure by reducing the potential dioxin-like toxicity and carcinogenicity (Abramowicz, 1995). Aroclor 1260 can be considered to be more representative of the PCBs found in biota (Ngui and Bandiera, 1999). One of the mechanisms by which PCBs may exert their promoting activity is by increasing hepatic oxidative stress (Glauert et al., 2008). Several studies have shown that PCBs increase lipid peroxidation in the liver (Fadhel et al., 2002). PCBs can also lead to oxidative DNA damage, in the form of 8-hydroxydeoxyguanosine (Oakley et al., 1996). a-Lipoic acid (LA) has gained considerable attention as an excellent antioxidant to reduce oxidative stress (Evans and Goldfine, 2000). Further, LA is fat- and water-soluble, which makes it effective against a broader range of free radicals. It has also been demonstrated recently that LA has ability to prevent hepatic steatosis in rat fed a long-term high-fat diet (Valdecantos et al., 2012). These beneficial effects make LA possess the potential abilities to improve nonalcoholic fatty liver diseases (Jung et al., 2012). The toxicity of most PCBs has been intensively studied. However, the published data describing liver toxicity in response to Aroclor 1260 exposure are limited and the mechanism of toxicity has not been completely characterized. The present study was aimed to investigate the mechanistic aspect of Aroclor 1260 induced hepatotoxicity and its protection by LA in adult rats.
experiments with animals were carried out according to the guidelines of the Institutional Animal Ethical Committee. The animals were randomly divided into six groups consisting of six animals each. Group I received vehicle and served as control. Group II received LA (35 mg/kg/day, dissolved in normal saline at alkaline pH, 7.8) and served as drug control group. Aroclor 1260 was dissolved in corn oil and given to rats by oral gavage at doses 20, 40, or 60 mg/ kg/day (Groups III, IV, and V, respectively). Group VI was pretreated with lipoic acid (35 mg/kg/day), 24 h before Aroclor 1260 (40 mg/kg/day) treatment. Treatment of animals with LA (Group II), Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V, respectively) and LA 1 Aroclor 1260 (40 mg/kg) (Group VI) were continued throughout the experiment for 15 consecutive days. The doses and duration of treatment were selected as per previous publications (Harris et al., 1993; Andric et al., 2000; Andric et al., 2003; Selvakumar et al., 2006).
Liver Function Tests Twenty four hours after the last treatment, blood samples were collected from the retro-orbital sinus, under ether anesthesia. Blood was allowed to clot for 30 min at (25 6 2 C). Then samples were centrifuged (2200 3 g for 10 min at 4 C) and supernatant serum was separated from the clot as soon as possible and stored at 220 C. Subsequently, serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were measured on a spectrophotometer using commercial diagnostic kit according to the recommendations of the manufacturer (Biodiagnostic, Cairo, Egypt). Total bilirubin (TB), total protein (TP), total albumin, total cholesterol (TC), triglycerides, and high-density lipoprotein (HDL) were measured in serum using commercial diagnostic kit according to the recommendations of the manufacturer (Biodiagnostic).
Oxidative Stress Status MATERIALS AND METHODS Reagents Aroclor 1260 and LA were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). All other chemicals are of analytical grade.
Animals and Treatment Adult male Wistar rats weighing 170 6 10 g were housed in clean polypropylene cages and maintained on a 12 h light/dark cycle and a temperature of 20–25 C with ad libitum access to food and water. For 7 days before the experiment, rats were handled daily for 5 min to acclimatize them to human contact and minimize their physiological responses to handling for subsequent protocols (Ma and Lightman, 1998). All the
Environmental Toxicology DOI 10.1002/tox
The animals were euthanized after blood collection. The liver was excised, weighed, and homogenized (10% w/v) in ice-cold 50 mM phosphate buffer (pH 7.4). The homogenate was centrifuged (4000 3 g for 10 min at 4 C) and the resulting supernatant was used for determination of oxidative stress status and apoptosis. Hydrogen peroxide (H2O2) production, lipid peroxidation, enzymatic activity of superoxide dismutase (SOD), and catalase (CAT) and non-enzymatic level of GSH were measured using colorimetric assay kits according to the manufacturer’s instructions (Biodiagnostic)
Caspases-3 & -9 Caspase-3 and -9 activities were measured in liver homogenate through cleavage of a colorless substrate specific for
Group II 59.67 6 5.13 117.5 6 7.89 161.83 6 5.34 895 6 31.47
Group I
63.83 6 5.85 120.17 6 6.49 162.83 6 6.91 913.33 6 36.7
a
72.83 6 3.31 * 132.67 6 5.85a* 176 6 5.62a* 996.67 6 41.31a*
Group III 74.5 6 4.37 * 138.17 6 6.49a** 179.17 6 7a** 1060 6 37.42a***
a
Group IV 77 6 6.2 ** 147.83 6 7.81a*** 180.67 6 8.48a** 1115 6 45.93a***
a
Group V
65.5 6 4.85b* 124 6 7.59b* 165.83 6 6.97b* 941.67 6 46.22b***
Group VI
0.3 6 0.03 101 6 5.1 63 6 5.06 6.48 6 0.62 4 6 0.37 51.83 6 3.97
0.31 6 0.05 102 6 4.86 67.83 6 5.71 6.3 6 0.65 3.98 6 0.33 50.67 6 4.8
Total bilirubin (TB) (mg/dl) Total cholesterol (TC) (mg/dl) Triglycerides (mg/dl) Total protein (TP) (g/dl) Total albumin (TA) (g/dl) HDL (mg/dl)
0.39 6 0.03* 115.17 6 8.47a* 77.67 6 5.24a* 5.27 6 0.48a* 3.32 6 0.34a* 42.83 6 3.31
Group III
Group V 0.42 6 0.04a** 125.5 6 8.12a*** 88.17 6 6.18a*** 5.02 6 0.58a** 3.08 6 0.36a** 38.17 6 6.77a**
Group IV 0.41 6 0.05a** 121.17 6 9.47a** 81.17 6 5.74a** 5.15 6 0.55a* 3.2 6 0.33a** 40.17 6 5.56a*
Doses of Aroclor 1260 (mg/kg/day)
0.33 6 0.05b* 106.83 6 5.46b* 69.33 6 4.72b** 6.28 6 0.5b* 3.83 6 0.34b* 49 6 4.24b*
Group VI
Abbreviation: LA, a-lipoic acid. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: a Group I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
Group II
Group I
Parameters
TABLE II. Effect of Aroclor 1260 and/or lipoic acid on serum biochemical parameters
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; LA, a-lipoic acid; LDH, lactate dehydrogenase. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: a Group I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
ALT (SGPT) (U/L) AST (SGOT) (U/L) ALP (U/L) LDH (U/L)
Serum Enzymes
Doses of Aroclor 1260 (mg/kg/day)
TABLE I. Effect of Aroclor 1260 and/or lipoic acid on the activities of serum ALT, AST, ALP, and LDH
LIPOIC ACID ATTENUATES AROCLOR 1260-INDUCED HEPATOTOXICITY
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and p < 0.01, respectively), AST (p < 0.05, p < 0.01, and p < 0.001, respectively) in a dose-related manner, ALP (p < 0.05, p < 0.01, and p < 0.01, respectively), and LDH (p < 0.05, p < 0.001, and p < 0.001, respectively) as compared to the corresponding control. Pretreatment with LA (Group VI) significantly suppressed (p < 0.05) the changes in the activity of ALT, AST, and ALP (p < 0.05) in addition to LDH (p < 0.001) induced by Aroclor 1260 (40 mg/kg/ day) treatment and normalized the activities of these enzymes as compared to Group IV (Table I).
Serum TB, TC, Triglycerides, TP, TA, and HDL Aroclor 1260 (20, 40, or 60 mg/kg) treatment (Groups III, IV, and V, respectively) significantly increased TB (p < 0.05, p < 0.01, and p < 0.01, respectively), TC (p < 0.05, p < 0.01, and p < 0.001, respectively), and triglycerides (p < 0.05, p < 0.01, and p < 0.001, respectively) as compared to the related control (Table II). The response of TC and triglycerides in response to Aroclor 1260 treatment
Fig. 1. Effect of Aroclor 1260 and/or lipoic acid on H2O2 production and lipid peroxidation in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/ day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). LA, alipoic acid. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
caspase-3 (Ac-DEVD-pNA) or caspase-9 (Ac-LEHD-pNA) releasing the chromophore, p-nitroaniline (pNA). The activities of caspases were measured at 405 nm. Assay was carried out according to the manufacturer’s instructions (Biodiagnostic).
Statistical Analysis Differences between obtained values (mean 6 SD, n 5 6) were compared by one way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test. A P value less than 0.05 was taken as a criterion for a statistically significant difference.
RESULTS Serum Activities of ALT, AST, ALP, and LDH The data showed that Aroclor 1260 (20, 40, or 60 mg/kg) treatment (Group III, IV, and V, respectively) caused a significant increase in the activity of ALT (p < 0.05, p < 0.05,
Environmental Toxicology DOI 10.1002/tox
Fig. 2. Effect of Aroclor 1260 and/or lipoic acid on the SOD and CAT activities in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/ kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/ day) 1 Aroclor 1260 (40 mg/kg/day). CAT, catalase; LA, alipoic acid; SOD, superoxide dismutase. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
LIPOIC ACID ATTENUATES AROCLOR 1260-INDUCED HEPATOTOXICITY
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(Group VI) suppressed H2O2 production (p < 0.01) and lipid peroxidation (p < 0.05) and normalized the activities of SOD, CAT, and GSH (p < 0.05) as compared to Group IV.
Caspases
Fig. 3. Effect of Aroclor 1260 and/or lipoic acid on GSH level in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/ day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/ day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). LA, a-lipoic acid. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01.
As shown in Figure 4(A,B), treatment with Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V) demonstrated significant increase in the activity of both caspase-3 (p < 0.05, p < 0.01, and p < 0.001, respectively) and caspase-9 (p < 0.05, p < 0.001, and p < 0.001, respectively) as compared to the related control. The increase in caspase-3 activity showed a dose-response manner. The increase in the activity of caspase-3 & -9 almost completely suppressed (p < 0.05 and 0.001, respectively) by pretreatment with LA (Group VI) as compared to Group IV.
DISCUSSION Liver is a vital organ that plays a pivotal role in metabolism and detoxification of various drugs as well as xenobiotics
was in a dose-related manner. Pretreatment with LA (Group VI) reverted the increase in TB (p < 0.05), TC (p < 0.05), and triglycerides (p < 0.01) to normal level as compared to Group IV (Table II). There were significant decrease in TP (p < 0.05, p < 0.05, and p < 0.01, respectively) and TA (p < 0.05, p < 0.01, and p < 0.01, respectively) in rats treated with Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V, respectively) as compared to the corresponding control (Table II). HDL did not show any significant change in response to Aroclor 1260 at a dose of 20 mg/kg (Group III), while presented significant decrease (p < 0.05 and p < 0.01, respectively) in response to treatment with Aroclor 1260 at doses 40 and 60 mg/kg (Groups IV and V, respectively) (Table II). Pretreatment with LA (Group VI) reverted the changes in TP, TA, and HDL (p < 0.05) to normal levels as compared to Group IV (Table II).
Liver Oxidative Stress Status Treatment with Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V) presented significant increase in both H2O2 production and lipid peroxidation (p < 0.05, p < 0.01, and p < 0.001, respectively) in a dose-related pattern as compared to the related control (Fig. 1A and B respectively). The activity of both SOD and CAT showed significant reduction (p < 0.05, p < 0.01 & p < 0.001 respectively) in response to Aroclor 1260 (20, 40 or 60 mg/kg) treatment (Groups III, IV & V) as compared to the corresponding control [Fig. 2(A,B)]). GSH level was significantly decreased (p < 0.05, p < 0.05, and p < 0.01, respectively) in response to Aroclor 1260 (20, 40, or 60 mg/kg) treatment (Groups III, IV, and V) as compared to the related control (Fig. 3). LA pretreatment
Fig. 4. Effect of Aroclor 1260 and/or lipoic acid on caspase-3 and 29 activities in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). LA, a-lipoic acid. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
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(Yang et al., 2010). However, it is the most common target organ for chemically induced injuries (Liu et al., 2013) Furthermore, serum enzymes, including ALT, AST, ALP, and LDH are marker enzymes for liver function and integrity (Adaramoye et al., 2008). These enzymes are usually elevated in acute hepatotoxicity or hepatocellular injury (Uzunhisarcikli and Kalender, 2011). Our study demonstrated a significant increase in serum activity of ALT, AST, ALP, and LDH in Aroclor 1260-treated rats. Elevated serum levels of these enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver (Sun et al., 2013). When the hepatocellular plasma membrane is damaged, the enzymes normally present in the cytosol are released into the blood stream. This can be quantified to assess the type and extent of liver injury (Ozturk et al., 2009; Ai et al., 2013; Liu et al., 2013). The increase in ALP and LDH is considered as a result of progressive liver necrosis (Bagchi et al., 1995; Mansour and Mossa, 2010). Severe hepatocellular death and liver dysfunction can lead to acute liver failure (Malhi et al., 2006). Also, it has been reported that the increased serum levels of these enzymes result from lipid peroxidation and increased H2O2 production which cause oxidative damage to the cell membrane (Yadav et al., 1997). Treatment with LA decreased the serum levels of ALT, AST, ALP, and LDH toward their normal value that is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage caused by Aroclor 1260 (Anusha et al., 2011). The restored level of these enzymes could be explained by the antioxidant effect of LA. On the other hand ALP is an indicator of pathological alteration in biliary flow (Ploa and Hewitt, 1989). Aroclor 1260 induced elevation of serum ALP is in line with high levels of serum bilirubin. Our results are in agreement with previous findings of Soudani et al. (2011) who suggested that the increase in serum bilirubin is a clear marker of hepatic dysfunction. Effective control of ALP and bilirubin levels in LA treatment group points towards an early improvement in the secretary mechanism of hepatocytes and indicating an important role of LA in protecting the integrity and functioning of tissues and cells. The liver is the site of cholesterol and triglycerides synthesis. Hyperlipidemia and hypercholesterolemia are reported as the major risk factors in life style related diseases such atherosclerosis and related cardiovascular complications including cerebral paralysis and myocardial infarction (Chovane`ıkova and Simek, 2001). The increased serum cholesterol level can be attributed to the effects of Aroclor 1260 on the permeability of liver cell membranes. Also, the obtained results of hyperlipidemia may be attributed to an increase in the synthesis of fatty acids in the liver or possibility due to incidence of liver cholestasis (Owings and Georgeson, 2000). Another cause of hyperlipidemia may be the abnormal activities of lipases enzymes which seem to be one of the chief factors
Environmental Toxicology DOI 10.1002/tox
responsible for the rise in serum total cholesterol (Hassan and Yousef, 2009). The total protein level is depressed in hepatotoxic conditions due to defective protein biosynthesis in liver (Clawson, 1989). In this study, Aroclor 1260-treated animals also exhibited significantly lower total protein and albumin levels than the control animals. Albumin most often transports or binds drugs or chemicals (Kalender et al., 2010). Albumin is synthesized by the liver and may decrease in individuals with liver function disorders. The changes of total protein level is based on function of albumin and globulin proportion, that could vary based on immunocompetence ‘‘status’’ of the animals or other pathophysiological condition (Petterino and Argentino-Storino, 2006). Further, the Aroclor 1260 intoxication may cause disruption and disassociation of polyribosomes on endoplasmic reticulum and thereby reducing the biosynthesis of protein. Treatment with LA restored proteins synthesis to normal level. It is well documented that a low level of HDL is indicative of high risk for cardiovascular diseases; an increase in HDL could potentially contribute to antiatherogenicity (Wilson et al., 1988; Assmann and Nofer, 2003). The lowered level of HDL and increased total cholesterol recorded in the serum of Arolcor 1260-treated rats revealed the severity of hepatopathy (Aniya et al., 2005). Pretreatment with lipoic acid significantly prevented or ameliorated this effect. Oxidative stress is attributed to either an increase in reactive oxygen species (ROS) generation and/or a decrease in the antioxidant defense mechanisms which lead to many degenerative diseases including a variety of hepatopathies (Hensley et al., 2000). The data presented here indicate that exposure of rat to Arolcor 1260 promoted increases in lipid peroxidation in the liver tissue and that treatment with LA significantly reduced its level. The increase in lipid peroxidation can most likely be ascribed to excessive ROS production, which could be related to antioxidant enzyme leakage (Yen et al., 2009; Shim et al., 2010; Zhang et al., 2013). ROS bind with polyunsaturated fatty acids of hepatic cell membranes initiating lipid peroxidation, with subsequent apoptosis or necrosis and increase lysosomal enzymes activities and liver damage (Chang et al., 2009; Ai et al., 2013; Ottu et al., 2013; Sinha et al., 2013). Therefore, Aroclor 1260 may induce conformational changes and whole cell deformity in the lipid bilayers of cell membranes, an effect that can lead to an increase in the membrane permeability and facilitates the passage of cytoplasmic enzymes outside the cells leading to the increase in the ALT, AST, ALP, and LDH activities in blood (Albukhari et al., 2009; Ai et al., 2013). Reactive oxygen species such as H2O2, superoxide anions, and hydroxyl radicals are generated under normal cellular conditions and are immediately detoxified by major scavenger enzymatic and nonenzymatic molecules (Lobo et al., 2010). However, excessive H2O2 production by Aroclor 1260 causes antioxidant imbalance and leads to lipid peroxidation and antioxidant depletion (Fadhel et al., 2002).
LIPOIC ACID ATTENUATES AROCLOR 1260-INDUCED HEPATOTOXICITY
Antioxidant enzymes including SOD and CAT are regarded as the first line of the antioxidant defense system against ROS generation during oxidative stress (Valko et al., 2007). CAT converts harmful H2O2 to harmless H2O, and SOD eliminates superoxide anions by producing H2O2 (Marklund and Marklund, 1974). Decrease in the activities of these enzymes alters the redox status of the cells. In the present study, the activity of the antioxidant enzymes SOD and CAT were significantly decreased. The antioxidant defenses were not able to effectively scavenge ROS, thus leading to lipid peroxidation. GSH is one of the main non-protein thiols and is a primary reductant that is present in cells (Yilmaz et al., 2006). GSH has the ability to protect cells from oxidative stress and plays a critical role in detoxification reactions by acting both as a nucleophilic scavenger of various undesirable compounds and their toxic metabolites and as a specific substrate for the glutathione peroxidase and glutathione-S-transferase enzymes (Sk and Bhattacharya, 2006). The decrease in GSH level during exposure to Aroclor 1260 may be due to the increased utilization of GSH, which can be converted into oxidized glutathione, and inefficient GSH regeneration. LA is an endogenous antioxidant, which can neutralize ROS as well as reducing the oxidized forms of other antioxidants due to its low redox potential (Sun et al., 2012). It was reported that LA may increase intracellular glutathione level (Islam, 2009) and glutathione peroxidase activity (Mantovani et al., 2003). LA is reported to be used as a therapeutic agent in a wide variety of conditions related to liver disease, such as including alcohol-induced damage, mushroom poisoning, metal toxicity, and CCl4 poisoning (Bustamante et al., 1998; Goraca et al., 2011). In the present study, treatment with LA may have increased the GSH level via increased GSH biosynthesis or by increasing the levels of other antioxidants. However, treatment with LA enhanced the antioxidant capacity, thus protecting the liver from Aroclor 1260-induced damage, as indicated by the maintenance of the antioxidant enzymes activities, decreased lipid peroxidation and increased the GSH level. Apoptosis, or programmed cell death, as well as the elimination of apoptotic cells are crucial factors in the maintenance of liver health (Dini, 1999; Manuela, 2001). Apoptosis involves a cascade of intracellular events, the process of which could not be stopped once be activated (Sun et al., 2012). Cytotoxic drugs and cellular stress activate the intrinsic mitochondrial apoptotic pathway (Kang and Reynolds, 2009). A character of apoptosis is the activation of caspase family members (Jiang et al., 2013). It is now well established that in most cell types, once cytochrome c is released into the cytosol, it triggers caspase-9 activation, results in the cleavage of caspase-3, and ultimately leads to the activation of the execution phase of apoptosis (Yin and Ding, 2003; Jiang and Wang, 2004). Our results showed that after Arolcor 1260 treatment, considerable elevation in caspase-3 and -9 activities were detected suggesting
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apoptotic effect. Apoptosis may contribute to the process of liver fibrosis (Shi et al., 1998; Lee et al., 2006). Several researchers report the anti-apoptotic effects and antioxidative properties of lipoic acid (Persson et al., 2001; Diesel et al., 2007; Duenschede et al., 2007). Duenschede et al. (2007) indicated that apoptotic hepatocyte numbers significantly decreased in LA treatment of hepatic ischemia/reperfusion injury. Our data support Duenschede et al.’s results that LA also reduces apoptosis by its antioxidative potential. In this study, we observed a significant decrease in caspase3 and -9 activities in group treated with LA. More importantly, LA inhibited the activation of molecular pathways of apoptosis. Since LA is a potent antioxidant which can scavenge ROS, the powerful regulatory effect of LA on the apoptotic pathway-related events was mediated by inhibiting the production and attenuating the activity of ROS (Sun et al., 2012). In summary, rats exposed to Aroclor 1260 showed impairment in liver function as evidenced by elevated serum levels of ALT, AST, ALP, and LDH activities, increased total bilirubin, total cholesterol, triglycerides while total protein, total albumin and HDL levels were significantly decreased. Aroclor 1260 induced H2O2 production, lipid peroxidation and concomitant decrease of antioxidant scavenging enzyme activities of SOD and CAT and GSH level. Further, Aroclor 1260 enhanced caspase-3 and -9 activities. Pretreatment with LA reverted these abnormalities to normalcy. In conclusion, Aroclor 1260 induced liver dysfunction, at least in part, by induction of oxidative stress. Apoptotic effect of hepatic cells is involved in Aroclor 1260-induced liver injury. LA could protect rats against Aroclor 1260-induced hepatotoxicity. The authors acknowledge with thanks DSR technical and financial support.
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Environmental Toxicology DOI 10.1002/tox
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Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
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Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
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Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, El-Madinah El-Munaworah, Saudi Arabia
Received 29 May 2014; revised 5 December 2014; accepted 7 December 2014 ABSTRACT: The present study was aimed to investigate the mechanistic aspect of Aroclor 1260-induced hepatotoxicity and its protection by lipoic acid. The adult male Albino rats were divided into six groups. Group I served as control. Group II received lipoic acid (35 mg/kg/day). Aroclor 1260 was given to rats by oral gavage at doses 20, 40, or 60 mg/kg/day (Groups III, IV, and V, respectively). Group VI was pretreated with lipoic acid (35 mg/kg/day) 24 h before Aroclor 1260 (40 mg/kg/day). Treatment in all groups was continued for further 15 consecutive days. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase activities and total bilirubin, total cholesterol, and triglycerides were significantly increased while total protein, total albumin, and high-density lipoprotein were significantly decreased. Hydrogen peroxide production and lipid peroxidation were significantly increased while superoxide dismutase and catalase activities and reduced glutathione (GSH) content was significantly decreased in liver. Caspase-3 & -9 activities were significantly increased in liver. Lipoic acid pretreatment significantly reverted all these abnormalities toward their normal levels. In conclusion, Aroclor 1260 induced liver dysfunction, at least in part, by induction of oxidative stress. Apoptotic effect of hepatic cells is involved in Aroclor 1260-induced liver injury. Lipoic acid could protect rats against AroC 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. clor 1260-induced hepatotoxicity. V Keywords: liver; Aroclor 1260; oxidative stress; apoptosis; lipoic acid
INTRODUCTION Polychlorinated biphenyls (PCBs) are ubiquitous, persistent organic pollutants and are a particularly problematic group of environmental contaminants. They comprise a class of 209 individual congeners which are numbered from an order Correspondence to: H. A. A. Aly; e-mail: [email protected] Contract grant sponsor: Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah. Contract grant number: 244/166/1433. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22101
of low to high chlorine substitutions (Mills et al., 2007). Due to their high lipophilicity they are mainly found in non-polar matrices such as lipids and adipose tissue or are attached to particulate matter such as sediments and dust (Giesy and Kannan, 1998). PCBs have been widely used in various applications, including dielectric fluids for capacitors and transformers, paints, adhesives, and pesticides because of their chemical and physical stability. Therefore, they have a strong tendency to bioaccumulate in our ecosystem (Yum et al., 2010). Their resistance to breakdown and their lipophilicity allows them to biomagnify in the food chain and persist in the environment (Hansen, 1987). PCBs are also well known as strong endocrine disruptors, acting as ligands
C 2014 Wiley Periodicals, Inc. V
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for steroid hormone receptors, such as the estrogen receptor (Yum et al., 2010). A number of these have structural and toxicological similarities with “dioxins” and elicit their effects via binding to the aryl hydrocarbon receptor (Roos et al., 2011). It is well known that a number of PCBs are potent liver toxicants (Safe, 1994). Arochlor 1260 shows the highest average percent weight of the chlorine and showing the lowest degradation rate by microorganism among PCB congeners (Yadav et al., 1995). Dechlorination of PCBs has been known to reduce the potential risk of PCB exposure by reducing the potential dioxin-like toxicity and carcinogenicity (Abramowicz, 1995). Aroclor 1260 can be considered to be more representative of the PCBs found in biota (Ngui and Bandiera, 1999). One of the mechanisms by which PCBs may exert their promoting activity is by increasing hepatic oxidative stress (Glauert et al., 2008). Several studies have shown that PCBs increase lipid peroxidation in the liver (Fadhel et al., 2002). PCBs can also lead to oxidative DNA damage, in the form of 8-hydroxydeoxyguanosine (Oakley et al., 1996). a-Lipoic acid (LA) has gained considerable attention as an excellent antioxidant to reduce oxidative stress (Evans and Goldfine, 2000). Further, LA is fat- and water-soluble, which makes it effective against a broader range of free radicals. It has also been demonstrated recently that LA has ability to prevent hepatic steatosis in rat fed a long-term high-fat diet (Valdecantos et al., 2012). These beneficial effects make LA possess the potential abilities to improve nonalcoholic fatty liver diseases (Jung et al., 2012). The toxicity of most PCBs has been intensively studied. However, the published data describing liver toxicity in response to Aroclor 1260 exposure are limited and the mechanism of toxicity has not been completely characterized. The present study was aimed to investigate the mechanistic aspect of Aroclor 1260 induced hepatotoxicity and its protection by LA in adult rats.
experiments with animals were carried out according to the guidelines of the Institutional Animal Ethical Committee. The animals were randomly divided into six groups consisting of six animals each. Group I received vehicle and served as control. Group II received LA (35 mg/kg/day, dissolved in normal saline at alkaline pH, 7.8) and served as drug control group. Aroclor 1260 was dissolved in corn oil and given to rats by oral gavage at doses 20, 40, or 60 mg/ kg/day (Groups III, IV, and V, respectively). Group VI was pretreated with lipoic acid (35 mg/kg/day), 24 h before Aroclor 1260 (40 mg/kg/day) treatment. Treatment of animals with LA (Group II), Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V, respectively) and LA 1 Aroclor 1260 (40 mg/kg) (Group VI) were continued throughout the experiment for 15 consecutive days. The doses and duration of treatment were selected as per previous publications (Harris et al., 1993; Andric et al., 2000; Andric et al., 2003; Selvakumar et al., 2006).
Liver Function Tests Twenty four hours after the last treatment, blood samples were collected from the retro-orbital sinus, under ether anesthesia. Blood was allowed to clot for 30 min at (25 6 2 C). Then samples were centrifuged (2200 3 g for 10 min at 4 C) and supernatant serum was separated from the clot as soon as possible and stored at 220 C. Subsequently, serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were measured on a spectrophotometer using commercial diagnostic kit according to the recommendations of the manufacturer (Biodiagnostic, Cairo, Egypt). Total bilirubin (TB), total protein (TP), total albumin, total cholesterol (TC), triglycerides, and high-density lipoprotein (HDL) were measured in serum using commercial diagnostic kit according to the recommendations of the manufacturer (Biodiagnostic).
Oxidative Stress Status MATERIALS AND METHODS Reagents Aroclor 1260 and LA were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). All other chemicals are of analytical grade.
Animals and Treatment Adult male Wistar rats weighing 170 6 10 g were housed in clean polypropylene cages and maintained on a 12 h light/dark cycle and a temperature of 20–25 C with ad libitum access to food and water. For 7 days before the experiment, rats were handled daily for 5 min to acclimatize them to human contact and minimize their physiological responses to handling for subsequent protocols (Ma and Lightman, 1998). All the
Environmental Toxicology DOI 10.1002/tox
The animals were euthanized after blood collection. The liver was excised, weighed, and homogenized (10% w/v) in ice-cold 50 mM phosphate buffer (pH 7.4). The homogenate was centrifuged (4000 3 g for 10 min at 4 C) and the resulting supernatant was used for determination of oxidative stress status and apoptosis. Hydrogen peroxide (H2O2) production, lipid peroxidation, enzymatic activity of superoxide dismutase (SOD), and catalase (CAT) and non-enzymatic level of GSH were measured using colorimetric assay kits according to the manufacturer’s instructions (Biodiagnostic)
Caspases-3 & -9 Caspase-3 and -9 activities were measured in liver homogenate through cleavage of a colorless substrate specific for
Group II 59.67 6 5.13 117.5 6 7.89 161.83 6 5.34 895 6 31.47
Group I
63.83 6 5.85 120.17 6 6.49 162.83 6 6.91 913.33 6 36.7
a
72.83 6 3.31 * 132.67 6 5.85a* 176 6 5.62a* 996.67 6 41.31a*
Group III 74.5 6 4.37 * 138.17 6 6.49a** 179.17 6 7a** 1060 6 37.42a***
a
Group IV 77 6 6.2 ** 147.83 6 7.81a*** 180.67 6 8.48a** 1115 6 45.93a***
a
Group V
65.5 6 4.85b* 124 6 7.59b* 165.83 6 6.97b* 941.67 6 46.22b***
Group VI
0.3 6 0.03 101 6 5.1 63 6 5.06 6.48 6 0.62 4 6 0.37 51.83 6 3.97
0.31 6 0.05 102 6 4.86 67.83 6 5.71 6.3 6 0.65 3.98 6 0.33 50.67 6 4.8
Total bilirubin (TB) (mg/dl) Total cholesterol (TC) (mg/dl) Triglycerides (mg/dl) Total protein (TP) (g/dl) Total albumin (TA) (g/dl) HDL (mg/dl)
0.39 6 0.03* 115.17 6 8.47a* 77.67 6 5.24a* 5.27 6 0.48a* 3.32 6 0.34a* 42.83 6 3.31
Group III
Group V 0.42 6 0.04a** 125.5 6 8.12a*** 88.17 6 6.18a*** 5.02 6 0.58a** 3.08 6 0.36a** 38.17 6 6.77a**
Group IV 0.41 6 0.05a** 121.17 6 9.47a** 81.17 6 5.74a** 5.15 6 0.55a* 3.2 6 0.33a** 40.17 6 5.56a*
Doses of Aroclor 1260 (mg/kg/day)
0.33 6 0.05b* 106.83 6 5.46b* 69.33 6 4.72b** 6.28 6 0.5b* 3.83 6 0.34b* 49 6 4.24b*
Group VI
Abbreviation: LA, a-lipoic acid. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: a Group I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
Group II
Group I
Parameters
TABLE II. Effect of Aroclor 1260 and/or lipoic acid on serum biochemical parameters
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; LA, a-lipoic acid; LDH, lactate dehydrogenase. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: a Group I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
ALT (SGPT) (U/L) AST (SGOT) (U/L) ALP (U/L) LDH (U/L)
Serum Enzymes
Doses of Aroclor 1260 (mg/kg/day)
TABLE I. Effect of Aroclor 1260 and/or lipoic acid on the activities of serum ALT, AST, ALP, and LDH
LIPOIC ACID ATTENUATES AROCLOR 1260-INDUCED HEPATOTOXICITY
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Environmental Toxicology DOI 10.1002/tox
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and p < 0.01, respectively), AST (p < 0.05, p < 0.01, and p < 0.001, respectively) in a dose-related manner, ALP (p < 0.05, p < 0.01, and p < 0.01, respectively), and LDH (p < 0.05, p < 0.001, and p < 0.001, respectively) as compared to the corresponding control. Pretreatment with LA (Group VI) significantly suppressed (p < 0.05) the changes in the activity of ALT, AST, and ALP (p < 0.05) in addition to LDH (p < 0.001) induced by Aroclor 1260 (40 mg/kg/ day) treatment and normalized the activities of these enzymes as compared to Group IV (Table I).
Serum TB, TC, Triglycerides, TP, TA, and HDL Aroclor 1260 (20, 40, or 60 mg/kg) treatment (Groups III, IV, and V, respectively) significantly increased TB (p < 0.05, p < 0.01, and p < 0.01, respectively), TC (p < 0.05, p < 0.01, and p < 0.001, respectively), and triglycerides (p < 0.05, p < 0.01, and p < 0.001, respectively) as compared to the related control (Table II). The response of TC and triglycerides in response to Aroclor 1260 treatment
Fig. 1. Effect of Aroclor 1260 and/or lipoic acid on H2O2 production and lipid peroxidation in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/ day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). LA, alipoic acid. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
caspase-3 (Ac-DEVD-pNA) or caspase-9 (Ac-LEHD-pNA) releasing the chromophore, p-nitroaniline (pNA). The activities of caspases were measured at 405 nm. Assay was carried out according to the manufacturer’s instructions (Biodiagnostic).
Statistical Analysis Differences between obtained values (mean 6 SD, n 5 6) were compared by one way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test. A P value less than 0.05 was taken as a criterion for a statistically significant difference.
RESULTS Serum Activities of ALT, AST, ALP, and LDH The data showed that Aroclor 1260 (20, 40, or 60 mg/kg) treatment (Group III, IV, and V, respectively) caused a significant increase in the activity of ALT (p < 0.05, p < 0.05,
Environmental Toxicology DOI 10.1002/tox
Fig. 2. Effect of Aroclor 1260 and/or lipoic acid on the SOD and CAT activities in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/ kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/ day) 1 Aroclor 1260 (40 mg/kg/day). CAT, catalase; LA, alipoic acid; SOD, superoxide dismutase. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
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(Group VI) suppressed H2O2 production (p < 0.01) and lipid peroxidation (p < 0.05) and normalized the activities of SOD, CAT, and GSH (p < 0.05) as compared to Group IV.
Caspases
Fig. 3. Effect of Aroclor 1260 and/or lipoic acid on GSH level in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/ day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/ day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). LA, a-lipoic acid. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01.
As shown in Figure 4(A,B), treatment with Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V) demonstrated significant increase in the activity of both caspase-3 (p < 0.05, p < 0.01, and p < 0.001, respectively) and caspase-9 (p < 0.05, p < 0.001, and p < 0.001, respectively) as compared to the related control. The increase in caspase-3 activity showed a dose-response manner. The increase in the activity of caspase-3 & -9 almost completely suppressed (p < 0.05 and 0.001, respectively) by pretreatment with LA (Group VI) as compared to Group IV.
DISCUSSION Liver is a vital organ that plays a pivotal role in metabolism and detoxification of various drugs as well as xenobiotics
was in a dose-related manner. Pretreatment with LA (Group VI) reverted the increase in TB (p < 0.05), TC (p < 0.05), and triglycerides (p < 0.01) to normal level as compared to Group IV (Table II). There were significant decrease in TP (p < 0.05, p < 0.05, and p < 0.01, respectively) and TA (p < 0.05, p < 0.01, and p < 0.01, respectively) in rats treated with Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V, respectively) as compared to the corresponding control (Table II). HDL did not show any significant change in response to Aroclor 1260 at a dose of 20 mg/kg (Group III), while presented significant decrease (p < 0.05 and p < 0.01, respectively) in response to treatment with Aroclor 1260 at doses 40 and 60 mg/kg (Groups IV and V, respectively) (Table II). Pretreatment with LA (Group VI) reverted the changes in TP, TA, and HDL (p < 0.05) to normal levels as compared to Group IV (Table II).
Liver Oxidative Stress Status Treatment with Aroclor 1260 (20, 40, or 60 mg/kg) (Groups III, IV, and V) presented significant increase in both H2O2 production and lipid peroxidation (p < 0.05, p < 0.01, and p < 0.001, respectively) in a dose-related pattern as compared to the related control (Fig. 1A and B respectively). The activity of both SOD and CAT showed significant reduction (p < 0.05, p < 0.01 & p < 0.001 respectively) in response to Aroclor 1260 (20, 40 or 60 mg/kg) treatment (Groups III, IV & V) as compared to the corresponding control [Fig. 2(A,B)]). GSH level was significantly decreased (p < 0.05, p < 0.05, and p < 0.01, respectively) in response to Aroclor 1260 (20, 40, or 60 mg/kg) treatment (Groups III, IV, and V) as compared to the related control (Fig. 3). LA pretreatment
Fig. 4. Effect of Aroclor 1260 and/or lipoic acid on caspase-3 and 29 activities in rat liver. Group I: Control (vehicle), Group II: LA (35 mg/kg/day), Group III: Aroclor 1260 (20 mg/kg/day), Group IV: Aroclor 1260 (40 mg/kg/day), Group V: Aroclor 1260 (60 mg/kg/day), and Group VI: LA (35 mg/kg/day) 1 Aroclor 1260 (40 mg/kg/day). LA, a-lipoic acid. Data are expressed as mean 6 SD (n 5 6). Statistical analysis (ANOVA) for differences from corresponding control: Comparisons were made between: aGroup I and Groups II–V; bGroup IV and Group VI. The symbols represent statistical significance from control where *p < 0.05; **p < 0.01; ***p < 0.001.
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(Yang et al., 2010). However, it is the most common target organ for chemically induced injuries (Liu et al., 2013) Furthermore, serum enzymes, including ALT, AST, ALP, and LDH are marker enzymes for liver function and integrity (Adaramoye et al., 2008). These enzymes are usually elevated in acute hepatotoxicity or hepatocellular injury (Uzunhisarcikli and Kalender, 2011). Our study demonstrated a significant increase in serum activity of ALT, AST, ALP, and LDH in Aroclor 1260-treated rats. Elevated serum levels of these enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver (Sun et al., 2013). When the hepatocellular plasma membrane is damaged, the enzymes normally present in the cytosol are released into the blood stream. This can be quantified to assess the type and extent of liver injury (Ozturk et al., 2009; Ai et al., 2013; Liu et al., 2013). The increase in ALP and LDH is considered as a result of progressive liver necrosis (Bagchi et al., 1995; Mansour and Mossa, 2010). Severe hepatocellular death and liver dysfunction can lead to acute liver failure (Malhi et al., 2006). Also, it has been reported that the increased serum levels of these enzymes result from lipid peroxidation and increased H2O2 production which cause oxidative damage to the cell membrane (Yadav et al., 1997). Treatment with LA decreased the serum levels of ALT, AST, ALP, and LDH toward their normal value that is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage caused by Aroclor 1260 (Anusha et al., 2011). The restored level of these enzymes could be explained by the antioxidant effect of LA. On the other hand ALP is an indicator of pathological alteration in biliary flow (Ploa and Hewitt, 1989). Aroclor 1260 induced elevation of serum ALP is in line with high levels of serum bilirubin. Our results are in agreement with previous findings of Soudani et al. (2011) who suggested that the increase in serum bilirubin is a clear marker of hepatic dysfunction. Effective control of ALP and bilirubin levels in LA treatment group points towards an early improvement in the secretary mechanism of hepatocytes and indicating an important role of LA in protecting the integrity and functioning of tissues and cells. The liver is the site of cholesterol and triglycerides synthesis. Hyperlipidemia and hypercholesterolemia are reported as the major risk factors in life style related diseases such atherosclerosis and related cardiovascular complications including cerebral paralysis and myocardial infarction (Chovane`ıkova and Simek, 2001). The increased serum cholesterol level can be attributed to the effects of Aroclor 1260 on the permeability of liver cell membranes. Also, the obtained results of hyperlipidemia may be attributed to an increase in the synthesis of fatty acids in the liver or possibility due to incidence of liver cholestasis (Owings and Georgeson, 2000). Another cause of hyperlipidemia may be the abnormal activities of lipases enzymes which seem to be one of the chief factors
Environmental Toxicology DOI 10.1002/tox
responsible for the rise in serum total cholesterol (Hassan and Yousef, 2009). The total protein level is depressed in hepatotoxic conditions due to defective protein biosynthesis in liver (Clawson, 1989). In this study, Aroclor 1260-treated animals also exhibited significantly lower total protein and albumin levels than the control animals. Albumin most often transports or binds drugs or chemicals (Kalender et al., 2010). Albumin is synthesized by the liver and may decrease in individuals with liver function disorders. The changes of total protein level is based on function of albumin and globulin proportion, that could vary based on immunocompetence ‘‘status’’ of the animals or other pathophysiological condition (Petterino and Argentino-Storino, 2006). Further, the Aroclor 1260 intoxication may cause disruption and disassociation of polyribosomes on endoplasmic reticulum and thereby reducing the biosynthesis of protein. Treatment with LA restored proteins synthesis to normal level. It is well documented that a low level of HDL is indicative of high risk for cardiovascular diseases; an increase in HDL could potentially contribute to antiatherogenicity (Wilson et al., 1988; Assmann and Nofer, 2003). The lowered level of HDL and increased total cholesterol recorded in the serum of Arolcor 1260-treated rats revealed the severity of hepatopathy (Aniya et al., 2005). Pretreatment with lipoic acid significantly prevented or ameliorated this effect. Oxidative stress is attributed to either an increase in reactive oxygen species (ROS) generation and/or a decrease in the antioxidant defense mechanisms which lead to many degenerative diseases including a variety of hepatopathies (Hensley et al., 2000). The data presented here indicate that exposure of rat to Arolcor 1260 promoted increases in lipid peroxidation in the liver tissue and that treatment with LA significantly reduced its level. The increase in lipid peroxidation can most likely be ascribed to excessive ROS production, which could be related to antioxidant enzyme leakage (Yen et al., 2009; Shim et al., 2010; Zhang et al., 2013). ROS bind with polyunsaturated fatty acids of hepatic cell membranes initiating lipid peroxidation, with subsequent apoptosis or necrosis and increase lysosomal enzymes activities and liver damage (Chang et al., 2009; Ai et al., 2013; Ottu et al., 2013; Sinha et al., 2013). Therefore, Aroclor 1260 may induce conformational changes and whole cell deformity in the lipid bilayers of cell membranes, an effect that can lead to an increase in the membrane permeability and facilitates the passage of cytoplasmic enzymes outside the cells leading to the increase in the ALT, AST, ALP, and LDH activities in blood (Albukhari et al., 2009; Ai et al., 2013). Reactive oxygen species such as H2O2, superoxide anions, and hydroxyl radicals are generated under normal cellular conditions and are immediately detoxified by major scavenger enzymatic and nonenzymatic molecules (Lobo et al., 2010). However, excessive H2O2 production by Aroclor 1260 causes antioxidant imbalance and leads to lipid peroxidation and antioxidant depletion (Fadhel et al., 2002).
LIPOIC ACID ATTENUATES AROCLOR 1260-INDUCED HEPATOTOXICITY
Antioxidant enzymes including SOD and CAT are regarded as the first line of the antioxidant defense system against ROS generation during oxidative stress (Valko et al., 2007). CAT converts harmful H2O2 to harmless H2O, and SOD eliminates superoxide anions by producing H2O2 (Marklund and Marklund, 1974). Decrease in the activities of these enzymes alters the redox status of the cells. In the present study, the activity of the antioxidant enzymes SOD and CAT were significantly decreased. The antioxidant defenses were not able to effectively scavenge ROS, thus leading to lipid peroxidation. GSH is one of the main non-protein thiols and is a primary reductant that is present in cells (Yilmaz et al., 2006). GSH has the ability to protect cells from oxidative stress and plays a critical role in detoxification reactions by acting both as a nucleophilic scavenger of various undesirable compounds and their toxic metabolites and as a specific substrate for the glutathione peroxidase and glutathione-S-transferase enzymes (Sk and Bhattacharya, 2006). The decrease in GSH level during exposure to Aroclor 1260 may be due to the increased utilization of GSH, which can be converted into oxidized glutathione, and inefficient GSH regeneration. LA is an endogenous antioxidant, which can neutralize ROS as well as reducing the oxidized forms of other antioxidants due to its low redox potential (Sun et al., 2012). It was reported that LA may increase intracellular glutathione level (Islam, 2009) and glutathione peroxidase activity (Mantovani et al., 2003). LA is reported to be used as a therapeutic agent in a wide variety of conditions related to liver disease, such as including alcohol-induced damage, mushroom poisoning, metal toxicity, and CCl4 poisoning (Bustamante et al., 1998; Goraca et al., 2011). In the present study, treatment with LA may have increased the GSH level via increased GSH biosynthesis or by increasing the levels of other antioxidants. However, treatment with LA enhanced the antioxidant capacity, thus protecting the liver from Aroclor 1260-induced damage, as indicated by the maintenance of the antioxidant enzymes activities, decreased lipid peroxidation and increased the GSH level. Apoptosis, or programmed cell death, as well as the elimination of apoptotic cells are crucial factors in the maintenance of liver health (Dini, 1999; Manuela, 2001). Apoptosis involves a cascade of intracellular events, the process of which could not be stopped once be activated (Sun et al., 2012). Cytotoxic drugs and cellular stress activate the intrinsic mitochondrial apoptotic pathway (Kang and Reynolds, 2009). A character of apoptosis is the activation of caspase family members (Jiang et al., 2013). It is now well established that in most cell types, once cytochrome c is released into the cytosol, it triggers caspase-9 activation, results in the cleavage of caspase-3, and ultimately leads to the activation of the execution phase of apoptosis (Yin and Ding, 2003; Jiang and Wang, 2004). Our results showed that after Arolcor 1260 treatment, considerable elevation in caspase-3 and -9 activities were detected suggesting
7
apoptotic effect. Apoptosis may contribute to the process of liver fibrosis (Shi et al., 1998; Lee et al., 2006). Several researchers report the anti-apoptotic effects and antioxidative properties of lipoic acid (Persson et al., 2001; Diesel et al., 2007; Duenschede et al., 2007). Duenschede et al. (2007) indicated that apoptotic hepatocyte numbers significantly decreased in LA treatment of hepatic ischemia/reperfusion injury. Our data support Duenschede et al.’s results that LA also reduces apoptosis by its antioxidative potential. In this study, we observed a significant decrease in caspase3 and -9 activities in group treated with LA. More importantly, LA inhibited the activation of molecular pathways of apoptosis. Since LA is a potent antioxidant which can scavenge ROS, the powerful regulatory effect of LA on the apoptotic pathway-related events was mediated by inhibiting the production and attenuating the activity of ROS (Sun et al., 2012). In summary, rats exposed to Aroclor 1260 showed impairment in liver function as evidenced by elevated serum levels of ALT, AST, ALP, and LDH activities, increased total bilirubin, total cholesterol, triglycerides while total protein, total albumin and HDL levels were significantly decreased. Aroclor 1260 induced H2O2 production, lipid peroxidation and concomitant decrease of antioxidant scavenging enzyme activities of SOD and CAT and GSH level. Further, Aroclor 1260 enhanced caspase-3 and -9 activities. Pretreatment with LA reverted these abnormalities to normalcy. In conclusion, Aroclor 1260 induced liver dysfunction, at least in part, by induction of oxidative stress. Apoptotic effect of hepatic cells is involved in Aroclor 1260-induced liver injury. LA could protect rats against Aroclor 1260-induced hepatotoxicity. The authors acknowledge with thanks DSR technical and financial support.
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